Studies is an experimental animal model have establishd that Mycobacterium bovis BCG and Mycobacterium smegmatis cell walls attached to oil droplets cause established tumors to regress and eliminate regional lymph node metastases when injected intralesionally. Components derived from these walls are currently in clinical use in veterinary and human medicine as immunotherapeutic agents for the treatment of cancer. The components of these cell walls that are effective in tumor regression include the arabinogalactan-peptidoglycan complex of the cell wall and a glycolipid constituent of its free lipid extract. Both components contain esterified mycolic acids, which have been found to be essential for effective therapy. Previous work has resulted in the complete structural characterization of the mycolic acids from M. smegmatis as well as the development of a general synthesis. The structural features of the arabinogalactan-peptidoglycan complex that are requisite for activity have not been identified. The overall objective of this proposed research is to structurally characterize the arabinogalactan-peptidoglycan complex and identify the structural features of that complex that are requisite for adjuvant activity. Structural characterization of the arabinogalactan will be accomplished by the reductive cleavage technique, a new procedure for polysaccharide structure determination developed in this laboratory during the past grant period. Several mycobacterial arabinogalactans will be examined by total reductive cleavage, furanose-specific reductive cleavage and partial reductive cleavage and the fragments so-formed will be separated and characterized by mass spectrometry and NMR spectroscopy. Identification of the structural features of the arabinogalactan-peptidoglycan complex that are requisite for adjuvant activity will be accomplished by establishing the structures of the peptidoglycan-derived glycopeptides and "peptido-glycolipids" formed as a result of "processing" of the bacterial cell wall by cultured macrophages. Studies during the past grant period have demonstrated that cultured murine macrophages rapidly catabolize peptidoglycan and release glycopeptides known to be adjuvant-active. Radiolabeling studies have also established that fragments of the peptidoglycan are retained in the macrophage membranes as covalent lipid derivatives. The isolation and structural characterization of these derivatives will be pursued in an effort to understand macrophage involvement in immunoregulatory functions.